A Single-Cell Study of a Highly Effective Hog1 Inhibitor for in Situ Yeast Cell Manipulation

Abstract

Abstract:We present a single cell study of a highly effective Hog1 inhibitor. For this application, we used sequential treatment of a Saccharomyces cerevisiae cell array, with the Hog1 inhibitor and osmotic stress. For this purpose, a four-inlet microfluidic chamber with controlled introduction of two different cell strains within the same experimental setting and a subsequent rapid switching between treatments was designed. Multiple cell strains within the same experiment is a unique feature which is necessary for determining the expected absent cellular response. The nuclear translocation of the cytosolic MAPK, Hog1, was monitored by fluorescence imaging of Hog1-GFP on a single-cell level. An optical tweezers setup was used for controlled cell capture and array formation. Nuclear Hog1-GFP localization was impaired for treated cells, providing evidence of a congenial microfluidic setup, where the control cells within the experiments validated its appropriateness. The chamber enables multiple treatments with incubation times in the order of seconds and the possibility to remove either of the treatments during measurement. This flexibility and the possibility to use internal control cells ensures it a valuable scientific tool for unraveling the HOG pathway, similar signal transduction pathways OPEN ACCESSMicromachines 2014, 5 82 and other biological mechanisms where temporal resolution and real time imaging is a prerequisite.

Roadmap for optical tweezers

“…As such, both intensity information (corresponding to the amount of the tagged protein in question) and localization, migration, or diffusion velocities of the fusion protein can be acquired. Incorporating fluorescence imaging and optical tweezers in the same optical setup also allows one to select individual cells to analyze from a cell population based on unique features, such as which reporter proteins they express [380].…”

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“…The total width and length of the channels are 2.0 cm and 3.2 cm, respectively, and the final PDMS system has total width and length of 3.9 cm and 4.6 cm, respectively, when using the described brass-frame design. This design has also been extended to devices with four inlet channels, enabling switching between two stress solutions (see, e.g., Gustavsson et al, 2015;Hamngren, Dinér, Grøtli, Goksör, & Adiels, 2014).…”

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